1. Field of the Invention
The present invention relates to a novel polydeoxyribonucleic acid having a genetic information of creatinase.
The invention also relates to a transformant having said polydeoxyribonucleic acid, and to the creatinase and a process for preparing the same through expression by the transformant of the genetic information of said polydeoxyribonucleic acid.
2. Description of the Background
Creatinase is a enzyme which catalyzes the reaction of creatine and water to produce urea and sarcosine. This substance has been known to exist in microorganisms belonging to genera of Pseudomonas Journal of General Microbiology, 14, 351,(1956)], Artherobactor [Molecular & Cellular Biochemistry, 3, 9, (1974)], Alcaligenes, . Penicilium (Japanese Patent Publication No. 7674/1981), Flavobacterium, Micrococcus, Colinebacterium (Japanese Patent Publication No. 8395/1977), Bacillus (Japanese Patent Publication No. 17465/1986), Actinobacillus (Japanese Patent Laid-open No. 67484/1981), and Acinetobactor (Japanese Patent Laid-open No. 67485/1981). Also, creatinase derived from Pseudomonas cutida by the use of genetic engineering technology has been reported.
Since creatinase is a hydrogenase having creatine as its substrate, it can be used for quantitation of creatine existing in a body fluid such as serum. It can also be utilized, through measurement of the quantity of creatine produced in the creatinine-creatinase creatine-producing system, for quantitation of various substances which are the substrates of enzymes affecting the creatine producing system as well as for the determination of enzymatic activities involved in the creatine-producing system. Creatinase is thus a very important reagent for use not only in laboratory experiment but also in clinical diagonosis.
Creatinase-producing microorganisms reported heretofore which are utilized without recourse to genetic engineering technique yield only a poor creatinase-producing efficiency so that the use of an enzyme-inducing substance such as creatine or the like which can help producing creatinase has been indispensable. This renders the cost of the creatinase production expensive. In addition, according to this method removal of other types of enzymes, such as creatininase or the like, which may be present together with creatinase, is very difficult. The cost of purification to obtain a high purity creatinase makes the overall production cost even higher. Thus, the use of these creatinase-producing microorganisms have not always been effective and convenient route for providing creatinase as reagents for abundant use in laboratory experiment or clinical diagonosis.
The creatinase derived from creatinase-producing microorganisms belonging to the genus of Bacillus also requires the addition of an expensive enzyme-inducing substance in the course of its preparation. In addition, there has been no report on the detailed chemical structure of the polypeptide constituting this enzyme.
The present inventors have undertaken extensive studies in order to improve the productivity of this creatinase, and have succeeded in obtaining the gene of this creatinase and further in clarifying its primary structural analysis. Further, the inventors have established a method for preparing the creatinase at a high productivity with the application of genetic engineering technique.
In addition, through analysis of the DNA sequence of the creatinase gene induced from the creatinase-producing microorganisms belonging to the genus of Bacillus the inventors have found that the enzyme possessed amino acid sequence and base sequence structures which are quite different from those of creatinase produced by conventionally known genetic engineering technique. The inventors also found that proteins obtained exhibited unexpectedly excellent creatinase activities. These findings have led to the completion of this invention.
The novel process for preparing creatinase according to this invention does not require the use of an expensive enzyme-inducing substance such as creatine in the course of creatinase production, and is thus a commercially advantageous process.